Process for obtaining antibodies utilizing heat treatment

ABSTRACT

This invention relates to a process for obtaining antibodies in soluble and correctly folded and assembled form. It comprises a step to raise the temperature at a time in the process selected to facilitate the subsequent isolation of soluble, correctly folded and assembled antibody, substantially free of other antibody-related material. The operating temperature may be raised at any stage in the microbial fermentation or eukaryotic cell culture, or at any stage during extraction and purification of the antibodies.

FIELD OF THE INVENTION

This invention provides an improvement to a process for obtainingantibodies in soluble, correctly folded and assembled form.

DESCRIPTION OF BACKGROUND ART

The rapid developments in recombinant DNA techniques have resulted inthe identification and isolation of many novel genes, some of knownfunction and some of unknown function. Invariably there is a need toexpress the gene in a heterologous cell system in order to producematerial for structure-function studies, diagnostic reagents such asmonoclonal or polyclonal antibodies and material for in vivo activitytesting and therapy.

Several alternative systems for the expression of foreign genes havebeen developed including systems based upon mammalian cells, insectcells, fungal cells, bacterial cells and transgenic animals or plants.The choice of expression system for a given gene depends upon the likelyfeatures of the encoded protein, for example any post-translationalprotein modifications needed for biological activity, as well as theobjective of the study. Other important considerations for theinvestigator are the facilities available and the time and cost involvedin generating the amounts of recombinant protein required.

The most widely used and convenient system for the production of foreignproteins remains that based on the prokaryote Escherichia coli. Theadvantages of this system comprise the ease of gene manipulation, theavailability of reagents including gene expression vectors, the ease ofproducing quantities of protein (up to a gramme in simple shake-flaskculture), speed and the high adaptability of the system to express awide variety of proteins.

Expression of any foreign gene in E. coli begins with the insertion of acDNA copy of the gene into an expression vector. Many forms ofexpression vector are available. Such vectors usually comprise a plasmidorigin of DNA replication, an antibiotic selectable marker and apromoter and transcriptional terminator separated by a multi-cloningsite (expression cassette) and a DNA sequence encoding a ribosomebinding site. The method of transcriptional regulation varies betweenthe various promoters now available (ptac, λpL, T7). The ptac and T7expression based systems are controlled by the chemical inducer IPTG,whilst the λ, promoters are controlled by a temperature switch.

A problem encountered with E. coli based expression systems is thedifficulty of producing material which is acceptable for therapeuticuse. The use of complex media, antibiotic selection and potentiallyhazardous inducers such as IPTG may potentially render products such asrecombinant antibody fragments produced by E. coli fermentationtechnology unacceptable to the regulatory authorities for clinicalapplications. Evidence demonstrating clearance of these agents from thefinal product must be provided in order to secure regulatory approval.Clearance of these agents, and especially demonstrating such clearance,is expensive. It is therefore desirable that an expression system shouldavoid the three above-mentioned problems.

A further problem is that proteins produced in bacterial cells are oftenprecipitated as insoluble aggregates within the bacterial cell. Thisproblem has been addressed in a number of ways in the prior art. Forexample, a large number of patent specifications teach solubilisation ofaggregates by the use of chaotropic denaturants and subsequentrenaturation. These procedures involve the use of expensive denaturingchemicals, are time-consuming and introduce chemical agents into theproduction process of which clearance demonstration will be required bythe regulatory authorities if the product is destined for clinical use.

An alternative approach has involved attempting to secrete theheterologous protein from the bacteria into the culture medium.

A number of recent advances have been made in bacterial proteinexpression, both relating to secretion and non-secretion systems. It hasbeen observed, by a number of groups working in this field, thatexpression of soluble protein products is favoured by culturing thebacteria at 30° C. or below.

For example, Cabilly (Gene, 85, p. 553-557, 1989) observed thatexpression of the Fd' fragment of an antibody directed againstcarcinoembryonic antigen (CEA) was improved at lower temperatures. Thisleads to a higher quantity of soluble heavy chain being recovered formthe bacteria after lysis of the cells. The Fd' fragment was expressedwith a complementary κ light chain fragment in order to allow theformation of Fab fragments. A greater yield of active, soluble Fabfragments was obtained at 21° C. and 30° C. than at 37° C.

Schein and Noteborn, Bio/Technology 6, p. 291-294, 1988, analysed theexpression of three proteins, human interferon-α2 (IFN-α2), humaninterferon-γ (IFN-γ) and murine Mx protein at 37° C. and at 23°-30° C.It was observed that proteins recovered from cell lysates were insolublewhen the bacteria were grown at 37° C. However, solubility was greatlyincreased by expression at 30° C. The formation of insoluble protein isdue to the aggregation of the heterologous polypeptide into inclusionbodies, a result of incorrect folding of the polypeptide chain.

The effect of temperature on proteins produced by secretion systems hasbeen shown to be similar. Chalmers et al, in Applied and EnvironmentalMicrobiology, 56 (1), p. 104-111, 1990, demonstrated that both humaninterferon and β-lactamase, both secreted proteins, produced inbacterial-cell culture were both more abundantly produced at 20° C. thanat 37° C. Furthermore, the incidence of inclusion bodies was reduced atthe lower temperature.

Chalmers et al conclude that for commercial production, as exemplifiedby chemostat experiments, culture at lower temperatures leads to moresoluble proteins being produced.

Antibodies and antibody fragments, especially chimeric, recombinant orhumanised derivatives thereof, are a class of proteins which it would beextremely desirable to be able to produce by recombinant DNA technology.By humanised antibodies, it is intended to refer to antibodies in whichthe constant regions are derived from human immunoglobulins, while atleast the complementarity determining regions (CDRs) of the variabledomains are derived from murine monoclonal immunoglobulins.

A number of improvements over natural immunoglobulins have beendocumented in the literature, which can only be put into practice byrecombinant DNA technology. For instance, the production of CDR-graftedantibodies having CDRs from murine antibodies coupled to human frameworkregions can only be undertaken using a recombinant expression system.Furthermore, such systems are extremely useful for the production ofantibody fragments which are not readily obtained by proteolyticcleavage, such as Fv fragments, and antibody fusions comprising aneffector or reporter molecule attached to the antigen binding molecule.

Recombinant antibody fragments, whether they be entire antibodies, Fab,Fab', F(ab')₂ or Fv fragments, consist of heavy and light chain dimers.A recombinant expression system should therefore be capable ofexpressing both heavy and light chain genes in such a manner as torender the individual peptides capable of self-assembly into the finalproduct. This has been a stumbling block for recombinant antibodyproduction, and indeed attempts have been made to solve the problem. Anexample of this is the production of "single chain" Fv fragments,wherein the heavy and light chain polypeptides are physically joinedtogether by a flexible linker group. These molecules avoid the problemsof chain association between free heavy and light chain polypeptides.

This system is not necessary, however, for the production of largerantibody fragments such as Fabs, which comprise heavy and light constantregion chains as well as heavy and light variable region chains. Thesefragments are large enough not to require coupling through a linker. Forsuch applications it is desirable to express heavy and light chainsseparately in the same cell.

In order to facilitate correct assembly of heavy and light chains ofantibody fragments, it is preferable to employ an expression system inwhich the chains are secreted into the periplasm of the host cell orinto the culture medium rather than precipitated into the cell asinclusion bodies.

Dual Origin vectors (DUOV), for example as described in our U.S. Pat.No. 5,015,573, have been found to be particularly suitable forexpressing antibody fragments. This has been shown to be the case,particularly when used in combination with protease-deficient bacterialhost cells (see our International Patent Specification WO89/02465). Thedual origin vector pAM1, (Wright et al., Gene, 49, p. 311, 1986), whichcomprises both pSC101 and colE1 replication functions, replicates at lowcopy number using the pSC101 replication functions at 30° C. At thistemperature, the colE1 replication functions, under the control of the λpR promoter, are tightly controlled by the c1857 repressor. Any foreignDNA inserted into the expression site of pAM1 is transcriptionallycontrolled by this being placed under the influence of the trp promoter,which is regulated by the host chromosomal trpR repressor.

The c1857 repressor is temperature sensitive, therefore increasing thetemperature of the growth medium above 34° C. leads to deregulation ofthe colE1 replication functions and increase in copy number from about 5per cell to several hundred per cell. This causes the host trpRrepressor to be titrated out, and transcription of foreign DNA from thetrp promoter can take place.

However, secretion of antibody fragments from host cells transformedwith DUOV vectors has not previously been attempted. In InternationalPatent Specification WO89/02465 we describe a process for the expressionof antibody fragments using DUOV vectors. However, the antibodyfragments are expressed intracellularly and are precipitated asinsoluble aggregates. These aggregates need to be solubilised by the useof chaotropic denaturants and/or other solubilisation techniques.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide aprocess for obtaining antibodies in soluble, correctly folded andassembled form. When antibody molecules are expressed in bacterial cellstransformed with a vector comprising DNA coding for said antibodies wehave found that SDS PAGE analysis of material secreted and exported intothe medium, material recovered from the periplasm and purified materialfrom either source, consists of four principal bands representingcorrectly folded and assembled antibody; partially degraded fragmentsthereof, and free heavy and light chain. We have now found that byintroducing an elevated temperature step it is possible to obtainsoluble, correctly folded and assembled antibody as revealed by thepresence of a single band on SDS PAGE.

According to the invention, therefore, there is provided an improvementto a process for obtaining antibodies in soluble, correctly folded andassembled form substantially free of other antibody related materialsaid improvement comprising the use in the process of a step to raisethe operating temperature to an elevated temperature at a time in theprocess selected to facilitate the subsequent isolation of soluble,correctly folded and assembled antibody substantially free of otherantibody related material.

Antibody which is `correctly folded and assembled` is shown by thepresence of a single band corresponding to the expected molecular weightfor assembled heavy and light chains on non-reducing SDS PAGE.

Other antibody related material will typically be free heavy and lightchain or a part thereof, partially degraded fragments of correctlyfolded and assembled antibody.

The process of the invention may be used for selectively isolatingantibodies in soluble, correctly folded and assembled form frommicrobial cell cultures or eukaryotic cell cultures.

The elevated temperature step may be introduced at any stage in theprocess for the production or isolation of antibodies, especiallyrecombinant antibodies. For example, the heat treatment may be carriedout at some stage during the expression phase of a fermentation or cellculture of a host cell transformed with at least one vector comprisingat least one heterologous DNA sequence coding for all or part of anantibody heavy and/or light chain, or some other stage of a fermentationor cell culture, or during the extraction and purification of theantibodies from the fermentation or cell culture. It may be carried outby heat treating the whole cells isolated from the fermentation or cellculture; by heat treating a homogenate of the whole cells; or byintroducing a heat treatment stage during the process of extraction andpurification of the antibodies from the fermentation or cell culture.

In a preferred embodiment the invention provides a process forselectively obtaining antibodies in soluble, correctly folded andassembled form wherein the operating temperature is raised to anelevated temperature step during the extraction and purification of theantibodies from a microbial cell culture or eukaryotic cell culture.

In a particularly preferred embodiment the invention provides a processfor selectively obtaining antibodies in correctly folded and assembledform from a solution containing a mixture of correctly folded andassembled antibody and other antibody related material comprising thesteps of 1. heating the said solution and 2. isolating the correctlyfolded and assembled antibody from the heated solution.

The correctly folded and assembled antibody may be isolated usingconventional techniques such as for example by homogenisation;clarification by filtration of centrifugation; precipitation, e.g. bytreatment with ammonium sulphate, polyethylene glycol or caprylic acid;affinity chromatography e.g. protein-A, protein-G, antigen-affinity oranti-IgG affinity chromatography; ion-exchange chromatography e.g. DEAEor hydroxyapatite; hydrophobic interaction chromatography or by gelfiltration. Methods for isolating and purifying antibodies are furtherdescribed in "Antibodies: A Laboratory Manual"; Ed Harlow, and DavidLane, published by Cold Spring Harbor Laboratories 1988.

These techniques may be used individually or in combination asappropriate depending on which stage in the process the operatingtemperature is raised to the elevated temperature.

The operating temperature is raised to an elevated temperaturepreferably at a stage in the extraction purification process prior tothe removal of cells or cell debris. We believe that the cells or celldebris assist in the removal of partially degraded and incorrectlyfolded antibodies for example during centrifugation.

Where the heat treatment step is carried out at the last stage in thepurification of the antibody it is desirable to mimic the cell and celldebris effect by providing a hydrophobic surface for denatured and/orincorrectly assembled antibodies to attach. The hydrophobic surface mayfor example be provided by using a batch hydrophobic chromatographymatrix, and adding this to the reaction mixture prior to heat treatment.Examples of such batch hydrophobic chromatography matrices am well knownin the art and include C₁₈ alkyl chains linked to a support matrix suchas sepharose, agarose, or silica e.g. butyl sepharose, phenyl sepharoseor octyl sepharose; or polymers such as cellulose or polystyrene.

In a particularly preferred embodiment the invention provides a processfor obtaining antibodies in correctly folded and assembled form from asolution containing a mixture of correctly folded and assembled antibodyand other antibody related material comprising the steps of 1. heatingthe said solution in the presence of batch hydrophobic chromatographymatrix material and 2. isolating the correctly folded and assembledantibody from the heated solution.

The solution will most conveniently be a partially purified solution,which may be for example heat treated at 40°-46° C. for 10-14 hours.

In a preferred embodiment, a homogenate of whole cells is heat treatedat 46° C. for 18 h prior to extraction and purification of the antibody.

The solution of antibody may for example be in a buffered solution usingbuffer salts such as Tris, acetate or phosphate. The pH of the antibodysolution may, for example be from pH2 to pH10 and will most especiallybe around neutral pH i.e. pH6-pH8. The ionic strength of the solution ofantibody may be varied using chaotropic salts such as ammonium sulphateor sodium sulphate. The solution of antibody may also be a pure aqueoussolution with no added solutes or an aqueous solution of a polar organicsolvent such as an alcohol e.g. ethanol.

The process according to the invention preferably selectively isolatesantibody molecules in soluble, correctly folded and assembled form.

Suitable examples of microbial host cells include bacteria such as grampositive bacteria or gram negative bacteria e.g. E. coli such as E. coliK12 strain W3110 or XL1 Blue or, yeast cells such as S. cerevisiaecells, and examples of eukaryotic cells include for example mammaliancells such as CHO cells and myeloma or hybridoma cell lines, forexample, NSO cells.

Where the operating temperature is raised to an elevated temperature atsome stage during the expression phase or some other stage of thefermentation or cell culture, heat treatment is preferably carried outfor a prolonged time. It may be, for example, for greater than 10 h,more preferably greater than 14 h and most preferably from 18-24 h.

Where the operating temperature is raised to an elevated temperaturestep during the extraction and purification of the antibodies from thefermentation or cell culture, this may be carried out for shorter timeperiods. For example, a solution containing a mixture of correctlyfolded and assembled antibody and other antibody related material may beflash treated at high temperatures or heat treated for up to 24 h,preferably for 10-18 h most preferably 12 h.

As used herein the term `elevated temperature` denotes temperatureswithin the range 34° C. to 60° C., more preferably 37° C. to 48° C. andis most preferably 45° C. or 46° C.

We have found that the process of the invention unexpectedly provides anefficient and convenient way of selecting out soluble, correctly foldedand assembled antibody.

The elevated temperature step may be introduced at the expression phaseof a fermentation or cell culture.

According to one embodiment of the process of the present invention,there is further provided a process for obtaining antibody molecules insoluble, correctly folded and assembled form from bacterial cell culturecomprising the steps of:

a) culturing a bacterial cell transformed with at least one expressionvector comprising a secretion signal sequence, an origin of replicationwhich is inducible from a repressed state at a repressive temperature,at which it replicates at a low copy number, to an induced state at anelevated permissive temperature, at which it replicates at a high copynumber, and a DNA coding sequence encoding all or part of an antibodymolecule comprising a light chain polypeptide and a heavy chainpolypeptide under the control of a promoter which is repressed when thevector is at a low copy number and of a secretion sequence, in a mediumat the repressive temperature at which the vector is maintained at a lowcopy number and recombinant gene expression is not induced;

b) raising the operating temperature of the culture medium to theelevated permissive temperature to induce replication of the vector tohigh copy number;

c) maintaining the operating temperature of the medium at the elevatedpermissive temperature; and

d) optionally collecting the antibody molecule product expressed intothe periplasm of the host cell or the culture medium.

It has now been found, surprisingly, that although a greater quantity ofprotein may be expressed and secreted from the host cell at therestrictive temperature, a greater proportion of this protein is solubleand correctly folded and assembled if the expression is carried out atthe elevated permissive temperature.

The restrictive temperature is preferably within the range of 10° to 33°C., while the elevated permissive temperature is within the range of 34°to 45° C. Advantageously, the restrictive temperature is 30° C. and theelevated permissive temperature is 37° C.

The inducible vector system for use in the present invention may be avector system in which vector copy number and expression of heterologousgene(s) is inducible by variation of the temperature at which the hostis cultured. For example, the expression system may comprise a runawayreplication vector of the type described in British Patent SpecificationGB-B-557774.

Preferably, however, the expression system comprises a dual originvector, for example as described in our British Patent SpecificationGB-A-2136814 or our U.S. Pat. No. 5,015,573. A dual origin vector is avector comprising two replication systems; a first origin of replicationresulting in a low copy number and stable inheritance of the vector, anda second, high copy number origin of replication at which replication isdirectly controllable as a result of replacement of or alteration by DNAmanipulation of the natural vector sequences which control replicationat said second origin.

The use of dual origin vectors has been found to be particularlyadvantageous for the expression of antibody products, due to theenhanced stability of the vectors.

A novel induction system which provides tightly regulated expressionwith medium to low copy number vectors in defined media which maintainsplasmid stability and allows the host cells to be cultured in theabsence of antibiotic selection is described in our copendingInternational patent application filed on even date herewith and derivedfrom British patent application number 9215550.6 filed 22nd Jul. 1992.This induction system is as follows: a host cell transformed with avector comprising a coding sequence under the control of an induciblepromoter which is repressed by a mature endogenous cellular repressor indefined medium is cultured under conditions such that the induciblepromoter is repressed, and expression of the heterologous protein isinduced by increasing the metabolic rate of the host cell therebydepleting the levels of the mature endogenous cellular repressor. Theincrease in metabolic rate is preferably brought about by switching thecarbon source, such as from glycerol to glucose.

The use of particularly stable expression vectors in defined medium inthe absence of antibiotic selection is described in our copendingInternational patent application filed on even date herewith and derivedfrom British patent application number 9215541.5 filed 22nd Jul. 1992.These stable expression vectors comprise one or more heterologous DNAsequences under the control of a regulatable promoter, an origin ofreplication and a transcriptional terminator.

The process for obtaining a soluble, correctly folded and assembledantibody described herein may be used in conjunction with the process ofeither or both of the above mentioned patent applications.

A vector for use in producing antibody molecules preferably comprisesheavy chain and light chain genes arranged with the light chain genelocated closer to the promoter such that it is transcribed first. It hasbeen observed that placing the light chain gene closer to the promoter,in such a manner that it is translationally coupled to the gene whichthe promoter is directly coupled to, and placing the heavy chain genedownstream from the light chain gene in such a manner that it does notbenefit from translational coupling, both cell viability and efficiencyof antibody secretion are enhanced.

In order to effect translational coupling, the natural coding sequenceof the bacterial gene whose promoter is being used in the expressionvector is altered in order to introduce a stop codon just before thebeginning of the sequence of the inserted heterologous gene. It ishypothesized that this causes ribosomes, which am efficiently assembledon the mRNA of the bacterial coding sequence, to become disengaged inthe close proximity of the translational start site of the heterologousmRNA. This favours the reassembly of the ribosomes on the heterologousmRNA, thus increasing the level of expression.

It is postulated that expression of an excess of heavy chains isdeleterious to the host cell. However, arranging the light chain genesuch that expression of light chain is favoured ensures an excess oflight chains in the cell, thus avoiding the problems associated withexcess heavy chain production. The arrangement of cistrons described isdesigned to favour the expression of an excess of light chains.

In a preferred embodiment of the vector for use in the invention,secretion of both heavy and light chain genes is directed by E. coliompA signal sequences. Preferably, the ompA translation initiationsignals are also included.

Advantageously, the ompA-antibody light chain fusion is translationallycoupled to the lacZ peptide translated from the tac promoter. This mayrequire the alteration of the ompA translation initiation sequence tointroduce a stop codon.

Advantageously, the culture medium used in the method of the inventionis a chemically defined medium. This allows the formulation of a processthe product of which is acceptable to the regulatory authorities, whichis highly desirable in the case of recombinant antibody products.

Examples of chemically defined medium are provided in Pirt S. J. (1975)"Principles of Microbe and Cell Cultivation", Blackwell ScientificPublications.

Preferably, the-defined medium used in the method of the invention doesnot contain antibiotic. It has been found that the dual origin vectorsused in the method of the invention are stable in the absence ofantibiotic selection. The stability of the plasmids is further improvedby favouring the expression of light chain, as described above.

In a further aspect the invention provides an antibody compositioncomprising soluble, correctly folded and assembled antibodysubstantially free of other antibody related material.

As used herein the term `substantially free of other antibody relatedmaterial` denotes that the correctly folded and assembled form of theantibody will be in excess of 90% more usually in excess of 95%, ofother antibody related material.

In a preferred embodiment of this aspect of the invention the antibodyis a recombinant antibody or fragment thereof.

The antibody molecules may comprise natural antibody molecules, chimericantibody molecules (the variable domains derived from one species andclass of antibody and remaining Ig sequences derived from anotherspecies or class of Ig), altered antibody molecules (variable Ig domainsplus an additional polypeptide sequence having a different, non-Igfunction, such as an enzyme or toxin), humanised antibody molecules andengineered antibody molecules (wherein the Ig amino acid sequence hasbeen altered from the natural sequence, e.g. by site-directedmutagenesis, with a view to altering a characteristic of the molecule,e.g. antigen binding specificity or affinity, for example as describedin Roberts et al., Nature, 238, 731-734, 1987). The antibody moleculesmay comprise suitable combinations of the above types of antibodymolecule.

Preferably, the antibody molecule is a humanised antibody moleculecomprising at least the CDRs of a non-human antibody attached to theframework of a human antibody.

More preferably, the antibody molecule is an antibody fragment. Forexample, the antibody molecule may be a Fab, Fab', (Fab')₂ or Fvfragment. Advantageously, it is a Fab or Fab' fragment.

The antibody molecules may have any desired antigen specificity. Forexample, the antibody molecules may have specificity for a cell-specificantigen, such as a tumour antigen, T cell marker, etc. Particularlypreferred are antibody molecules which have specificity fortumour-associated antigens such as CEA and TAG72. Chimeric A5B7antibodies and antibody fragments are described in our copendingInternational patent application WO 92/01059.

Also preferred are antibodies having specificity for the epitoperecognised by murine monoclonal antibody A33 as described in ourcopending British patent application number 9225853.2 filed 10th Dec.1992. Particularly preferred are humanised and chimeric forms of A33,and most particularly preferred are Fab' fragments thereof.

The antibodies may be site-specific antibodies such as tumour-specificor cell surface-specific antibodies, suitable for use in in vivo therapyor diagnosis, e.g. tumour imaging. Examples of cell surface-specificantibodies are anti-T cell-antibodies, such as anti-CD3, and CD4 andadhesion molecules, such as CR3, ICAM and ELAM. The antibodies may havespecificity for interleukins (including lymphokines, growth factors andstimulating factors), hormones and other biologically active compounds,and receptors for any of these. For example, the antibodies may havespecificity for any of the following: Interferons α, β, γ or δ, IL1,IL2, IL3 or IL4, etc., TNF, GCSF, GMCSF, EPO, hGH, or insulin, etc.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is now described in the following examples, with referenceto the following figures in which:

FIG. 1 is a diagram of dual origin vectors;

FIG. 2 is a photograph of an agarose gel electrophoresis experiment ofplasmid preparations derived from bacterial cultures transfected withplasmids comprising heavy and light chain genes in the orders cLc-cFd'and cFd'-cLc;

FIG. 3 is a photograph of a western blot which compares the yields ofantibody product from DUOV vectors transfected with cLc-cFd' andcFd'-cLc gene constructs;

FIG. 4 shows the comparative induction profiles of a dual origin vectorat 37° C. and 38° C.;

FIG. 5 shows relative product accumulation with varying inductionprofiles;

FIG. 6a is a western blot showing the different Fab' species obtained(a) under reducing conditions and (b) under non-reducing conditions; and

FIG. 6b is a western blot showing the different Fab' species obtainedunder non-reducing conditions;

FIG. 7 is a western blot comprising the quantity of correctly foldedFab' obtained by various methods.

FIG. 8 shows a western immunoblot of cell extracts after incubationovernight at a range of temperatures.

    ______________________________________                                                lane extract at                                                       ______________________________________                                                1     4° C.                                                            2    30° C.                                                            3    43° C.                                                            4    44° C.                                                            5    46° C.                                                            6     4° C.                                                            7    30° C.                                                            8    43° C.                                                            9    44° C.                                                            10   46° C.                                                    ______________________________________                                    

FIG. 9(a) shows western immunoblots of cell extracts

lane 1: A33 humanised Fab' extraction at 30° C.

lane 2: A33 humanized Fab' extraction at 46° C.

lanes 3-7 show cell lysates prepared by lysosyme treatment of cellpellets post incubation at 46° C.

(b) shows western immunoblots of cell extracts

lane 1 A5B7 humanised Fab' extraction at 30° C.

lane 2 A5B7 humanized Fab' extraction at 46° C.

(c) A5B7 humanised Fab'

lanes 1-7 show extracts made by lysosyme lysis post heat treatment at46° C.

FIG. 10 shows western immunoblot of cell extracts made at 30° C. (lanes1-8) and 46° C. (lanes 10-17).

FIG. 11 shows western immunoblot of cell lysates prepared by breaking ina French pressure cell. The extract was then incubated at either 40° C.or 46° C. Samples were taken at regular time intervals and stored onice. Lanes 1-6 incubation at 40° C. Lanes 7-11 incubation at 46° C.

FIG. 12a shows SDS PAGE anaylsis of Fab' samples extracted from cells byTris/EDTA treatment and purified by prosepA affinity chromatography. Thesamples were not heat treated.

FIG. 12b shows SDS PAGE analysis of a Fab' sample extracted from cellsby Tris/EDTA treatment and purified by prosepA affinity chromatography.The sample was heat treated.

FIG. 12(c) shows SDS PAGE analysis of Fab' samples extracted by lysosymelysis of cells and purified by prosepA affinity chromatography.

lane 1 non heat treated sample

lane 2 heat treated sample

FIG. 13 is a Western immunoblot of prosepA purified Fab' from apreparation not subjected to heat treatment prior to purification.

lane 1 unincubated Fab' prep

lane 2 Fab' prep incubated alone at 46° C. for 12 h

lane 3 Fab' prep incubated at 46° C. for 12 h in the presence of phenylsepharose.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

CONSTRUCTION OF HEAVY AND LIGHT-CHAIN FUSIONS

A5B7 heavy and light chain sequences cLc (chimeric light chain) and cFd'(chimeric Fd' heavy chain fragment) were isolated as described inInternational patent application WO 92/01059. These were fused to the E.coli secretion sequence omp A and termed ompA-cLc and ompA-cFd', asdescribed in International patent application WO 92/01059. The sequenceswere inserted into pSK (Stratagene) as described in WO 92/01059 toproduce pSKompA-cLc and pSKompA-cFd'.

pMRR026 was constructed by removing the DNA sequence coding for ompA-cLcas an Xho I-Sma I fragment from pSKompA-cLc, and inserting it into pSP73(promega Corp.) at the Sal I and Pvu 2 sites. pMRR027 was constructed asdescribed in WO 92/01059.

pAM1 (see FIG. 1) is a dual origin vector which replicates at low copynumber using the pSC101 replication functions at 30° C. (Wright et al.,Op. Cit.). At this temperature the colE1 replication functions, underthe control of the lambda pR promoter, are tightly controlled by the cl857 repressor, and the target gene under the control of the trp promoteris tightly controlled by the chromosomal trpR repressor. Because thec1857 repressor is temperature sensitive increasing the temperature toabove 37° C. leads to amplification of copy number from approximately5/cell to several hundreds/cell. This amplification titrates out the trprepressor, reducing the relative concentration thereof below a leveleffective to cause repression, and results in high level expression ofthe target gene. To construct a pAM1 derivative expressing the A5B7chimeric Fab' the ompA-cLc and ompA-cFd' fusions were each first clonedas Xhol-EcoRI fragments from pSKompA-cLc and pSKompA-cFd' into theSal1-EcoR1 gap of pAM1, to give plasmids pMRR030 and pMRR031. The EcorIfragment of pMRR027 carrying ompA-cFd' was then cloned into the EcoRIsite of pMRR030 to give pMRR032 (see FIG. 1) with the order trppromoter-cLc-cFd'. Similarly the EcoRI fragment of pMRR026 carrying theompA-cLc was cloned into EcoRI site of pMRR031 to give plasmid pMRR033,with the order trp promoter-cFd'-cLc.

pMRR032 and pMRR033 were transformed into strains XL1 Blue and W3110 forexpression studies in shake flasks and fermenters. Initial shake flaskexperiments on strains carrying pMRR033 suggested that this plasmid,like the other plasmids with this gene order, were structurallyunstable, and no further expression experiments were performed on them(see FIGS. 2 and 3).

FIG. 2 is an agarose gel showing preparations of pQ9Kan-cLc-cFd' andpQ9Kan-cFd'-cLc plasmids.

lane 1: λ-Hind III markers;

lanes 2 to 11: plasmid preparations from strain W3110 transformed withpQ9Kan-cLc-cFd' (A5B7);

lanes 12 to 16: plasmid preparations from strain W3110 transformed withpQ9Kan-cFd'-cLc (A5B7).

In lanes 2 to 11, the intact plasmid can be seen just above the 4.4 Kbmarker and no degradation products are apparent.

In lanes 12 to 16, on the other hand, degradation products may be seenat about 2.1 and 4 Kb.

FIG. 3 is a Western blot of culture supernatants and cell extracts fromcell lines transformed with DUOV vectors transformed with cLc-cFd' andcFd'-cLc genes.

lane 12: culture supernatant of XL1B transfected with pMRR032(cLc-cFd');

lanes 13 and 14: culture supernatants of XL1B transfected with pMRR033(cFd'-cLc).

lane 15: cell extract of XL1B transfected with pMRR032;

lanes 16 and 17: cell extract of XL1B transfected with pMRR033.

The cLc-cFd' gene order can be seen to give much higher yields ofproduct.

Because previous studies with the dual origin vector had suggested thatthe exact induction temperature is crucial inductions were carried outin 1.5 l fermenters on XL1Blue/pMRR032 at both 37° C. and 38° C. tooptimise the induction temperature for these plasmids. These twoinduction temperatures gave significantly different growth profiles andyields. Cultures induced at 37° C. continued to grow post induction fortwo to three hours longer than those induced at 38° C., with a resultanthigher titre of Fab' product appearing in the medium. Fab' yields wereagain estimated by immunoblotting, this method suggesting the yield at37° C. to be 5-10 mg/l.

FIG. 4 shows the relative amounts of pMRR032 DNA in samples throughthese fermentations. It suggests that induction at 38° C. resulted in arapid increase in plasmid copy number to an approximately constant valuewhile induction at 37° C. gives a slower but more prolonged increase inplasmid copy number which eventually exceeds the plateau observed at 38°C.

FIG. 4 is an agarose gel of plasmid DNA prepared from cell samples fromfermentations of XL1 Blue (pMRR032) induced at 37° C. or 38° C.

In FIG. 4 lane identification is as follows:

lane 1 λ Hind III markers

lanes 2-7 samples taken 10, 11, 13, 14, 16 and 17 hours after inductionat 37° C.

lanes 8-13 samples taken 10, 11, 13, 14, 16 and 17 hours after inductionat 38° C.

FERMENTATION PROCESS DEVELOPMENT USING CHIMERIC A5B7 Fab'

Temperature for induction, time at induction temperature before returnto 30° C., temperature ramp to induction and complex nitrogen feed wereall varied. Product titres were assessed by CEA-binding ELISAs on thecrude culture supernatants, with the results shown in FIG. 5.Amplification of copy number in defined medium proved to be lower thanpreviously observed for complex medium but was nevertheless sufficientto support the maximum rate of product accumulation. Fermentationsinduced at 37° C. or 38° C. and maintained at these temperatures for onehour accumulated more product than those induced at higher temperaturesor for longer periods. With these short inductions at 37° C. productcontinued to accumulate up to the point of harvest, 20 hours afterinduction. The other fermentation profiles gave shorter productaccumulation phases. The data are consistent with the view that maximumyield will result from partial induction to give a relatively low butsustainable level of expression compatible with the maximum rate oftranslocation, signal cleavage or maintenance of an unfoldedconformation in the cytoplasm.

The Fab' product appearing in the culture supernatants gave four majorbands on non-reducing PAGE immunoblotted with a polyclonal anti-humanFab antibody (FIG. 6). FIG. 6 shows an SDS-PAGE immunoblot of E. coliW3110 (pMRR032) cultured supernatants. Crude culture supernatant sampleswere run on a 12% polyacrylamide gel and probed with an anti-human IgGFab' polyclonal-HRP conjugate. On reducing PAGE the cross-reactingmaterial appeared as a single band with the same mobility as A5B7 Fab'made in mammalian cells. Different fermentations, but not different timeof harvesting, were found to give different relative proportions of thefour bands. Induction and subsequent growth till harvesting at 37° C.gave a predominance of the correctly assembled Fab' (see FIG. 7).

In FIG. 7 samples were electrophoresed on a 12% non-reducingpolyacrylamide gel then probed with an anti-human IgG Fab'polyclonal-HRP conjugate.

lanes: 1-2 pMRR32 (cA5B7 Fab' in d.o.v.) induced and maintained at 37°C.;

lanes 3-4 pMRR028 (cA5B7 Fab' in pACtac);

lanes 5-6 pMRR044 (gL1gH1 A5B7 Fab' in pACtac);

lanes 7-8 pMRR045 (gL1gH2 A5B7 Fab' in pACtac).

All pACtac plasmids were induced with 200 μM IPTG.

PRODUCTION OF ANTIBODY Fab' FRAGMENTS OF SINGLE BANDED QUALITY (ASDETERMINED BY SDS PAGE ELECTROPHORESIS) FROM E. coli EXPRESSION SYSTEMS

This invention describes a method for the production of antibody Fab'fragments largely free from contamination by partially degraded Fab' andfree heavy or light chains. The method comprises the use of an elevatedtemperature phase during the induction stage of the fermentation. Thework described here demonstrates that the elevated temperature phase maybe used after harvesting the fermentation during an extraction phase andresults in similar single banded quality product.

It is reported widely in the literature that, in general terms, lowerculture temperatures favour the production of soluble, correctly foldedrec DNA products in E. coli. Therefore, by delaying the period ofelevated temperature to the product extraction and recovery phases thefermentation may be carried out under conditions which are optimised forproduction of active Fab'. The partially degraded Fab' and free lightand heavy chains which accumulate under such conditions may be removedas described above by heat treatment after the fermentation.

The production of multibanded Fab' consisting of intact correctlyassembled Fab', two predominant degradation products and free light andheavy chains has been observed in:

Two separate expression hosts

At least 4 separate Fab' fragments

Fermentations using two different plasmids and induction systems

Therefore it appears that this problem is not restricted to anindividual expression system or protein.

The choice of 46° C. as the incubation temperature for the production ofsingle banded Fab' from a cell extract containing multibanded Fab' wasmade by testing a range of temperatures up to 46° C. The materialproduced after incubation at 46° C. was the highest quality.Temperatures above 46° C. were not tested. A western immunoblot of cellextracts incubated at a range of temperatures up to 46° C. is shown inFIG. 8, the samples were run reduced and non reduced to reveal the rangeof assembled and partially degraded Fab'. This set of extractions wasprepared by incubating the intact cells overnight in Tris HCl buffer 100mM pH 7.4 containing EDTA 10 mM.

FIG. 8 shows a western immunoblot of cell extracts of humanised A33 Fab'produced by resuspending cells harvested by centrifugation in Tris HClbuffer pH 7.4 100 mM containing 10 mM EDTA and incubating overnight at arange of temperatures.

lanes 1-5 samples were reduced with 2MCE

lanes 6-10 were run non-reduced.

lanes 1 and 6 were loaded with an extraction made at 4° C., 2 and 7 at30° C., 3 and 8 at 43° C., 4 and 9 at 44° C. and

lanes 5 and 10 at 46° C.

The bands labeled on the non-reduced side are:

    ______________________________________                                        1              Assembled intact Fab'                                          2 and 3        Partially degraded Fab'                                        4              Intact heavy and light chains                                  ______________________________________                                    

The titre of protein obtained in the 5 extracts was determined by a dyebinding assay kit (Pierce) and found to be 1.06, 0.7, 0.66, 0.28 and0.26 g/l for extracts made at 4°, 30°, 43°, 44° and 46° C. respectively.Therefore, while the quality of the Fab' material found in the extractsimproved with temperature the total protein in the extract was reducedby a factor of up to 4.

Range of fragments to which temperature treatment is applicable.

Extracts made from cells expressing humanised A33 Fab' and those madefrom cells expressing humanised and chimeric A5B7 Fab' have been shownto contain Fab' predominantly of the assembled intact molecular weightpost heat treatment. Prior to heat treatment these extracts containedfour main bands of similar intensity when visualised on a Westernimmunoblot.

Shown in FIG. 9 are extraction samples of A5B7 and A33 humanised Fab'expressed in E. coli strain W3110. The extractions were made with andwithout heat treatment at 46° C.

Humanised A33 Fab' may be constructed and expressed in E. coli asdescribed in British patent application number 9225853.2 filed 10th Dec.1992 and initial British patent application filed on even date herewith(ref. PA 345).

Heat treatment of periplasmic extracts and cell lysates.

The cell extracts described above were made by incubating cells inbuffer containing EDTA (10 mM) and resulted in a selective release ofperiplasmic proteins. Cell lysis by enzymatic or mechanical means wasfound to release more active Fab' than any selective extraction system.The unheated cell lysates were found to contain similar profiles of Faband degradation products to those found in Tris HCl/EDTA extracts. Celllysates could be heat treated to remove the partially degraded Fab andfree heavy and light chain found in the soluble fraction. The heattreatment phase could be used pre or post breakage/lysis with similaraffect. Shown in FIG. 9 are samples of cell extracts post heattreatment, these samples show that the Fab' is predominantly in theassembled intact form.

FIG. 9 shows Western immunoblots of cell extracts made by incubation inTris/EDTA buffer.

FIG. 9a shows A33 humanised Fab'.

lane 1 extraction made at 30° C.

lane 2 extraction made at 46° C.

lane 3-7 show cell lysates prepared by lysozyme treatment of cellpellets post incubation at 46° C.

FIG. 9b shows A5B7 humanised Fab'

lane 1 extraction made at 30° C.

lane 2 extraction made at 46° C.

FIG. 9c shows A5B7 humanised Fab'

lanes 1-7 show extracts made by lysozyme lysis post heat treatment at46° C.

Heat treatment of cell pellets sampled at regular intervals during afermentation

FIG. 10 shows a western immunoblot of cell extracts made by incubationin Tris HCl/EDTA buffer at 30° C. (lanes 1-8) and 46° C. (lanes 10-17).The samples loaded represent a time course across a fermentationexpressing humanised A33 Fab'.

Predominantly single banded material is shown in the samples extractedat 46° C. and 4 principal bands are shown in the samples extracted at30° C. Therefore, the partially degraded Fab' and free light and heavychains were found to be present when Fab' was first detected in theperiplasmic extract and samples taken throughout the fermentation couldbe converted to the predominantly single banded form.

Time course of heat treatment at 46° C.

Experiments were carried out to determine the incubation periods at 46°C. required to obtain predominantly intact single banded Fab' from amixture of degradation products, intact Fab' and free light and heavychain. These experiments tested two procedures:

1. The intact cells were incubated at 46° C. with samples being removedat appropriate intervals and stored on ice. All samples were then lysedand analysed by western immunoblotting and activity ELISA.

2. An aliquot of cell suspension was lysed then incubated at 46° C.Samples were taken at appropriate time intervals and analysed by westernimmunoblotting and activity ELISA.

Analysis of the samples by western immunoblotting

The quality of material as determined by Western immunoblotting wasfound to be best in the 20 h incubation sample at 40° C. and in the 10 hsample taken from the 46° C. incubation. The western immunoblot (FIG.11) shows no evidence of loss of the intact Fab' during eitherincubation, indeed the final samples show a clearer more intense Fab'band. This apparent increase in Fab' may be an artifact resulting frommore efficient transfer of the protein during blotting in the latersamples which are known to contain less total protein. The ELISAresults, in contrast to the western immunoblot, show a reduction intitre of material binding to the antigen on the solid phase. This mayhave resulted from some of the incorrectly assembled or partiallydegraded Fab' binding to the antigen and being detected in the assay.

FIG. 11 shows a Western immunoblot of cell lysates (expressing humanisedA33 Fab') prepared by breaking in a French pressure cell. The extractwas then incubated at either 40° C. or 46° C. Samples were taken atregular time intervals and stored on ice. Lanes 1-6 incubation at 40° C.Lanes 7-11 incubation at 46° C.

Recovery of Fab' proteins by Protein A purification pre and post heattreatment

The pattern of intact Fab', degradation products and free heavy andlight chains visualised by western blotting of samples pre and post heattreatment was found to be similar to that recovered by protein Apurification and analysis of column eluates by SDS PAGE and coomassieblue staining. Preparations of predominantly intact single banded Fab'from initial starting mixtures of Fab', degraded Fab' and free heavy andlight chains were obtained from both Tris HCl/EDTA periplasmic extractsand cell lysates. Shown in FIGS. 12(a)-(c) are samples of periplasmicextracts and cell lysates (expressing humanised A33 Fab') with and withand without heat treatment (FIGS. 12(a)-(c) lanes 1 to 5 periplasmicextracts non heat treated and lane 6 heat treated, FIG. 12(c) lane 1cell lysate non heat treated and lane 2 heat treated).

FIG. 12a shows SDS PAGE gels of Fab' samples extracted from cells byTris/EDTA treatment and purified by prosepA affinity chromatography.

lanes 1-5 non-heat treated

lane 6 heat treated.

FIG. 12(c) shows SDS PAGE gel of Fab' samples extracted by lysozymelysis of cells and purified by prosepA affinity chromatography.

lane 1 non heat treated

lane 2 heat treated.

Recovery of Single Banded Fab from Purified Material ContainingPartially Degraded Fab and Free Heavy and Light Chain

Material purified from cultures which had not been subjected to heattreatment has been shown to consist of four principal Fab associatedbands. Single banded (correctly assembled and intact) material may berecovered from purified four banded material by incubation at 46° C. inthe presence of Phenyl Sepharose. The partially purified material is ina solution of a citrate buffer pH2 in which the pH has been raised topH7 by the addition of Tris.

FIG. 13 shows a Western immunoblot of prosepA purified humanised A5B7Fab' from a preparation not subjected to heat treatment prior topurification.

lane 1 unincubated Fab' prep

lane 2 Fab' prep incubated alone at 46° C. for 12 h.

lane 3 Fab' prep incubated at 46° C. for 12 h in the presence of phenylsepharose.

The invention is described above by way of example only, and numerousmodifications of detail will be apparent to those skilled in the artwhich fall within the scope of the appended claims.

METHODS FOR THE EXPRESSION OF ANTIBODY FRAGMENTS IN E. coli USING THEDUAL ORIGIN AND pAC tac EXPRESSION VECTORS

1. Storage of Strains

2. Revival of Cultures and Inoculum Preparation

3. Media

4. Shake Flask Culture and Induction Procedures

4.1 Host strain W3110 with pAC tac vector

4.2 Host strain W3110 with dual origin vector

5. Fermentor Culture and Induction Procedures

5.1 Host strain W3110 with pAC tac vector

5.2 Host strain W3110 with dual origin vector

6. Periplasmic Extraction Procedures

1. STORAGE OF STRAINS

A single colony from a freshly transformed plate was streaked out on anLA plate containing the appropriate antibiotic selection. A singlecolony from this plate was used to inoculate a 250 ml Edenmeyer flaskcontaining 40 ml LB medium+appropriate antibiotic selection (dual originvector: ampicillin, pAC tac vector: chloramphenicol). This flask wasincubated at 30° C. and 250 RPM in an orbital incubator until theculture reached an optical density (OD 600 nm) of 2 (mid exponentialgrowth phase) taking approximately 8 h to reach this point.

Aliquots (750 μl) of this culture were mixed with 250 μl sterileglycerol solution (50% v/v in H₂ O) in a 2 ml sterile ampoule (Sterlin).These glycerol stocks were stored at -70° C. without controllingfreezing rate.

2. REVIVAL OF STRAINs AND INOCULUM PREPARATION

Inocula for all experiments were prepared from frozen glycerol stocks inLB medium containing Cm or Amp as appropriate, the seeding density wasusually 300 μl glycerol stock per litre LB. Inoculum cultures, grown inEdenmeyer flasks (1 L containing 200 ml medium) incubated at 30° C. and250 RPM in an orbital shaker were used when an OD 600 nm of 3 had beenattained (normally 12-16 h). Fermentors and shake flasks were seededwith 5-10% volumes of inoculum.

3. MEDIA

3.1 LA: Luria Agar

LA Cm: LA+chloramphenicol 25 μg/ml

LA Amp: LA+ampicillin 25 μg/ml

3.2 LB: Luria Broth

LB Cm and LB Amp both 25 μg/ml

3.3 YEGLY

    ______________________________________                                        Component             g/L                                                     ______________________________________                                        Glycerol              20.0                                                    (NH.sub.4).sub.2 SO.sub.4                                                                           7.0                                                     NaH.sub.2 PO.sub.4.2H.sub.2 O                                                                       6.24                                                    Yeast extract (Difco) 40.0                                                    SM6 trace elements    10 ml/L                                                 Antifoam solution (1)% mazu DF843)                                                                  1 ml/L                                                  ______________________________________                                    

This formulation was made up to 1 L with deionised water,

3.4 SM6

    ______________________________________                                        Component             g/L                                                     ______________________________________                                        (NH.sub.4).sub.2 SO.sub.4                                                                           5                                                       NaH.sub.2 PO.sub.4    6.24                                                    Trace element solution (SM6)                                                                        10 ml/L                                                 Antifoam solution (10% mazu DF843)                                                                  1 ml/L                                                  ______________________________________                                    

This formulation was made up to 0.96 L with deionised water, Where thecarbon source used was glycerol this was added to a concentration of 20g/L prior to autoclaving. Where glucose and or lactose were used thesewere added post sterilisation as 50% solutions (sterilised byautoclaving) to final concentrations of 20 g/L.

SM6 Trace element solution

    ______________________________________                                        Component    g/L 100x stock solution                                          ______________________________________                                        NaOH         15.0                                                             EDTA         60.0                                                             MgSO.sub.4.7H.sub.2 O                                                                      20.0                                                             CaCl.sub.2.6H.sub.2 O                                                                      5.0                                                              ZnSO.sub.4.4H.sub.2 O                                                                      2.0                                                              MnSO.sub.4.4H.sub.2 O                                                                      2.0                                                              CuSO.sub.4.5H.sub.2 O                                                                      0.5                                                              CoCl.sub.2.6H.sub.2 O                                                                      0.095                                                            FeSO.sub.4.7H.sub.2 O                                                                      10.0                                                             H.sub.3 BO.sub.3                                                                           0.031                                                            Na.sub.2 MoO.sub.4                                                                         0.002                                                            ______________________________________                                    

Each component was dissolved individually in deionised water and addedto the bulk solution in the sequence shown to a final volume of 1 L.

3.5 SM6A

    ______________________________________                                        Component             g/L                                                     ______________________________________                                        (NH.sub.4)SO.sub.4    5.0                                                     NaH.sub.2 PO.sub.4    6.24                                                    KCl                   3.87                                                    MgSO.sub.4.1H.sub.2 O 0.56                                                    Citrate               4.0                                                     SM6A Trace Element solution                                                                         10 ml/L                                                 Antifoam (Mazu DF843 10% in H.sub.2 O)                                                              1.0 ml/L                                                ______________________________________                                    

Made up to 0.95 L with deionised water.

SM6A Trace element solution

    ______________________________________                                        Component    g/L 100x stock solution                                          ______________________________________                                        Citrate      100.0                                                            CaCl.sub.2.6H.sub.2 O                                                                      5.0                                                              ZnSO.sub.4.4H.sub.2 O                                                                      2.0                                                              MnSO.sub.4.4H.sub.2 O                                                                      2.0                                                              CuSO.sub.4.5H.sub.2 O                                                                      0.5                                                              CoSO.sub.4.6H.sub.2 O                                                                      0.4                                                              FeCl.sub.3.6H.sub.2 O                                                                      9.67                                                             H.sub.3 BO.sub.3                                                                           0.03                                                             NaMoO.sub.4  0.02                                                             KCl          74.5                                                             ______________________________________                                    

Made up to 1 l with deionised water. Components were added in the ordershown and were allowed to dissolve completely prior to the addition ofthe next salt.

Defined media were brought to pH 6.95 using 3.6M NH₄ OH afterautoclaving.

Carbon sources for defined media were as described in the fermentationmethods section.

All media were sterilised by autoclaving at 121° C. for 20 min.

Glucose and Lactose were autoclaved separately as 50% solutions (w/v) inH₂ O and added to cultures as described in the fermentation methodssection. Prior to autoclaving, conc H₂ SO₄ (100 μl per litre) was addedto glucose solutions.

Glycerol for feeding during fermentations was autoclaved neat or as a50% w/v solution in H₂ O.

Casamino acids (Difco, 200 g/l solution in H₂ O sterilised byautoclaving) were added to give a final concentration of 20 g/L wheredescribed.

4. SHAKE FLASK CULTURE AND INDUCTION PROCEDURES

4.1 Host Strain W3110 with pAC tac Expression Vector

Shake flask cultures were made in 250 ml Erlenmeyer baffled flaskscontaining 40 ml YEGLY medium seeded with 4 ml inoculum prepared asdescribed in section 2. Cultures were incubated at 30° C. and 250 RPM inan orbital incubator. Induction of product expression was obtained at OD600 nm=5 by adding a 40 μl aliquot of IPTG (200 mM, freshly preparedaqueous solution sterilised by filtration). Cultures induced at an OD600 of 2.5 produced higher yields for certain fragments than thoseinduced at 5. Addition of IPTG to cultures which had reached OD's of 6or greater and had moved into the decline phase of growth did not induceproduct expression.

Culture supernatants were harvested by centrifugation 12 h postinduction with IPTG.

4.2 Host Strain W3110 with the Dual Origin Expression Vector

Cultures were grown as described in Section 4.1. Induction of productexpression was achieved by transferring flasks to an orbital incubatorpre equilibrated at 40° C. when cultures had reached an OD of 5.

Culture supernatants were harvested by centrifugation 12 h postinduction by temperature switching.

5. FERMENTOR CULTURE AND INDUCTION PROCEDURES

5.1 Host Strain W3110 with pAC tac Expression Vector

5.1.1 Complex medium fermentations

Fermentations were made in YEGLY medium inoculated at a seeding densityof 5% with the culture described in section 1. The culture pH wascontrolled at 7.0±0.05 by addition of 2M NaOH or 2M H₂ SO₄. Temperaturewas maintained at 30° C. and dissolved oxygen tension (DOT) wascontrolled at a value >10% air saturation by automatic control ofagitator speed. Aeration was set at 0.75 v/v/min. Oxygen utilisationrates and carbon dioxide evolution rates were determined from exhaustgas analysis performed by mass spectrometry. Product formation wasinduced by adding IPTG as a filter sterilised 1000× stock solution to afinal concentration of 200 μM when the culture OD had reached 5.

Fermentations were run with and without chloramphenicol (25 μg/ml), norequirement for antibiotic selection in the fermentation medium has beendemonstrated.

Culture supernatants were harvested 12 h post induction bycentrifugation 4200 RPM rmax 240 mm (1-2 l fermentations) or bytangential flow filtration (15 l and 150 l fermentations). Clarificationof broths was superior with tangential flow filtration (TFF).

5.1.2 Defined Medium Fermentations

Fermentations were made in medium SM6 or SM6A, glucose was used as theinitial carbon and energy source for all fermentations and was addedafter medium sterilisation to a concentration of 20 g/l. Culture pH wasbrought to and maintained at 6.95 by the addition of 3.6M NH₄ OH, or 2MH₂ SO₄. DOT was maintained above 10% by control of agitator speed,culture temperature was maintained at 30° C. throughout thefermentation.

IPTG inductions: Cultures were induced with IPTG (final concentration200 μM) at OD 600 nm=40. Cultures induced with IPTG were fed glucose asrequired, (either in response to OUR or as predicted by an approximateyield of 1 OD/g glucose/1).

Lactose inductions: Induction of product expression was also obtained byswitching the carbon source to lactose from glucose. Glucose was fed tosupport the culture to an OD of approximately 30 (an accumulativeaddition of 30 g/l). Lactose feeding was started at an OD ofapproximately 25, as with glucose, lactose was then fed (normally asindividual shots of 50% lactose to a concentration of 10 g/l culture) asrequired. The 50% lactose solution was held in a water bath at 55° C.after autoclaving to prevent crystallisation.

Cultures induced by IPTG were harvested 20 h post induction. Culturesinduced by lactose feeding were harvested 24-30 h after the switch fromglucose to lactose utilisation.

Where casamino acids were added to defined medium fermentations theseadditions were made 3 h post induction.

5.2 Host Strain W3110 with the Dual Origin Expression Vector

5.2.1 Complex medium fermentations

These fermentations were made as described in section 5.1.1 except thatinduction of product formation was achieved by increasing the culturetemperature when the OD had reached 20. A temperature switch to 37° C.from 30° C. and holding the culture at 37° C. was used.

5.2.2 Defined medium fermentations

These fermentations were made as described in section 5.1.2 except thatglycerol was used throughout as the carbon and energy source (startingconcentration 20 g/L). Medium SM6A could only be used with citratereduced to 1 g/L total.

Fermentations run using glucose as the carbon source resulted ininduction of product expression prior to temperature switching and inthe absence of plasmid amplification.

Cultures were fed glycerol as required, again in response to the onlineOUR data or as predicted by an approximate yield of 1 OD/g glycerol/L.

Fermentations were harvested 12 h post temperature induction bycentrifugation or TFF. Where inductions did not arrest growth (normally4-6 h post temperature shift) it was not possible to maintain the DOTabove 10% air saturation, these cultures were allowed to become oxygendepleted and harvested.

We claim:
 1. A process to facilitate the isolation of soluble,correctly-folded antibody molecules comprising subjecting a preparationcomprising soluble, correctly-folded antibody molecules and partiallydegraded or incorrectly folded antibodies to a step to raise theoperating temperature to an elevated temperature within the range of 34°C. to 60° C. in order to facilitate the removal of the partiallydegraded or incorrectly folded antibodies.
 2. A process according toclaim 1 wherein the preparation comprising soluble, correctly-foldedantibody molecules and partially degraded or incorrectly foldedantibodies is the product of a fermentation comprising culturing a hostcell transformed with an exression vector which encodes at least part ofan antibody light chain and an expression vector encoding at least partof an antibody heavy chain, such that at least some of the light chainand heavy chain molecules are secreted and combine to form soluble,correctly-folded and assembled antibody.
 3. A process according to claim2 wherein said step to raise the operating temperature is carried out at40° C. to 60° C. for a period of up to 18 hours.
 4. A process accordingto claim 2 wherein the preparation subjected to said step to raise theoperating temperature comprises disrupted cells.
 5. A process accordingto claim 2 wherein said step to raise the operating temperature iscarried out during the expression phase of the fermentation.
 6. Aprocess according to claim 2 wherein the preparation subjected to saidstep to raise the operating temperature further comprises a batchhydrophobic chromatography matrix.
 7. A process according to claim 5comprising the steps of:a) culturing a bacterial cell transformed withat least one expression vector comprising an origin of replication whichis inducible from a repressed state at a repressive temperature, atwhich it replicates at a low copy number, to an induced state at anelevated permissive temperature, at which it replicates at a high copynumber, and a DNA coding sequence encoding all or part of an antibodymolecule comprising a light chain polypeptide and a heavy chainpolypeptide under the control of a promoter which is repressed when thevector is at a low copy number and of a secretion sequence, in a mediumat the repressive temperature at which the vector is maintained at a lowcopy number and recombinant gene expression is not induced; b) raisingthe operating temperature of the culture medium to the elevatedpermissive temperature to induce replication of the vector to high copynumber; c) maintaining the operating temperature of the medium at theelevated permissive temperature and d) optionally collecting theantibody molecule product expressed into the periplasm of the host cellor the culture medium.
 8. A process according to claim 2, wherein saidantibody molecule is a natural, humanised or chimeric antibody or afragment thereof.
 9. A process according to claim 2, further comprisingthe step of isolating the soluble, correctly folded antibody molecules.10. A process according to claim 7, wherein in steps (b) and (c), saidelevated permissive temperature is 40°-60° C.
 11. A process according toclaim 10, wherein in step (c), said elevated permissive temperature ismaintained for a period of 10 to 18 hours.
 12. A process according toclaim 5, wherein said antibody molecule is a natural, humanised orchimetic antibody or a fragment thereof.